oxLDL-induced ROS generation and activation of gp91^phox/NOX2 requires PKC. (A) Washed human platelets incubated with apyrase, indomethacin, and EGTA were treated with PP2 and PP3 and then with LDL before being lysed with ice-cold lysis buffer.²⁶  Lysates were immunoblotted for phosphorylated PKC substrates. (i) Blots are representative of at least 3 experiments using different blood donors. (ii) Densitometric analysis of the blots. *P < .05 compared with oxLDL alone. (B) Washed human platelets (5 × 10⁷ platelets/mL) were incubated with BIMI or BIMV (both 10 µmol/L) for 20 minutes at 37°C or left untreated (control) for 20 minutes at 37°C, incubated with a fluorescent superoxide detection probe for 30 minutes at 37°C, and adhered to slides coated with nLDL or oxLDL (50 µg/mL). Images are representative of 3 independent experiments with separate blood donors. Scale bar, 20 µm. (C) Washed human platelets were incubated with apyrase, indomethacin, and EGTA to prevent secondary signaling. In addition, they were also incubated with PP2/PP3 (20 µmol/L); U73122 (5 µmol/L); BIMI, BIMV, and Ro31-8220 (10 µmol/L); FA6-152; or a control IgG (both 5 µg/mL), then lysed with ice-cold lysis buffer.²⁶  p47^phox was then immunoprecipitated (IP) and immunoblotted (IB) using an antibody against phosphorylated PKC substrates. (i) Blots are representative of at least 3 experiments using different blood donors. (ii) Densitometric analysis of the blots. *P < .05, **P < .01 compared with oxLDL alone. (D) Washed human platelets were incubated with nLDL or oxLDL (50 µg/mL) for 15 minutes at 37°C, then diluted 1:1 with cell fractionation buffer (320 mmol/L sucrose, 4 mmol/L N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, and 0.5 mmol/L Na₃VO₄, protease inhibitor cocktail [pH 7.4]). The suspensions were immediately snap-frozen in liquid nitrogen and lysates separated into soluble (S) and particulate (P) fractions by ultracentrifugation. Proteins from each fraction were separated by SDS-PAGE and immunoblotted for p47^phox, p22^phox, CD36, and Syk. (i) Blots re representative of at least 3 experiments using different blood donors. (ii) Densitometric analysis of the blots. *P < .05 compared with oxLDL alone.